The present invention relates to methods and materials for GABA production in cells and living organisms. More particularly, the invention relates to genetic transformation of organisms, preferably plants, with genes that encode proteins that catalyze the conversion of putrescine to GABA. GABA is known to function as a neurotransmitter in animals. In plants, increased levels of GABA are associated with increased tolerance to environmental stress.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference in their entirety for all that they disclose, and for convenience are referenced in the following text by reference number and are listed by reference number in the appended bibliography.
GABA in Response to Stress
In plants, GABA levels increase in response to a variety of stresses (1-4), but the biological significance of the rapid accumulation remains unknown (1-6). Several physiological roles for GABA synthesis in plants in response to stress have been proposed including: to maintain cytosolic pH (7, 8), to serve as an alternative store for carbon or nitrogen (N; (9), to deter insect feeding (10-12), or to serve as a signaling molecule (4-6, 13, 14).
Although the physiological role of GABA synthesis in plants in response to stress is not well defined, it has been clearly demonstrated that GABA is required for plant development and protection against biotic and abiotic stress (10-12), and that proper GABA levels, either through the regulation of biosynthesis (6, 15) or catabolism (5, 13), is required for normal plant growth and development (5, 13) and for stress survival (5). Furthermore, exogenous application of GABA results in increased plant growth and development. Elevated levels of GABA in plants may confer multiple agronomic benefits: increased plant growth and development (158, 4); increased tolerance to abiotic stresses, including drought (159), salinity (160), flooding (161), heat (162), freezing (163, 25), limited nutrient availability (164); and increased tolerance to biotic stresses, such as insect feeding (12, 165) and nematode infestation (12). GABA may function to alter nitrogen and/or carbon metabolism (1, 9, 14, 166, 167).
Metabolic Pathways that Affect GABA Accumulation in Plants
There are three known metabolic pathways that affect, or regulate, GABA levels in plants (FIG. 1). The first pathway is via the decarboxylation of glutamate by the enzyme glutamate decarboxylase (GAD or glutamic acid decarboxylase (16-18). The second pathway is via the GABA shunt (13, 19). All the components, enzymes and genes, of the two pathways have been demonstrated to exist in higher plants. A third pathway, which is associated with the catabolism of polyamines, and known to exist in bacteria, was reported over 20 years ago to exist in plant tissue (20, 21).
GABA Production by GAD
GABA accumulation in plants upon exposure to stress has been attributed to stimulation of GAD activity (1, 3, 22-25). GAD activity is controlled (23) by the binding of calcium and calmodulin to a 22-25 amino acid region at the carboxy-terminus of the protein, called a calmodulin-binding domain (26). The calmodulin-binding domain functions as an autoinhibitory domain to deactivate the GAD enzyme (15), which is located in the cytoplasm (10, 19).
GABA Shunt
Another way to control GABA levels is by regulating the breakdown or catabolism through enzymes in the GABA shunt. GABA, synthesized in the cytoplasm by GAD, is then transported into mitochondria where it is catabolized by enzymes in the GABA shunt (19). GABA is converted into succinate semialdehyde by pyruvate-dependent GABA transaminase (GABA-T) (13, 27). Succinate semialdehyde is catabolized into succinate by succinate semialdehyde dehydrogenase (SSADH) for use in the tricarboxylic acid (TCA) cycle (5, 28, 29). Succinate semialdehyde can also be converted into gamma-hydroxybutyrate by gamma-hydroxybutyrate dehydrogenase (30).
GABA Production Through Polyamine Catabolism
The catabolism of polyamines into GABA has been documented in plants (20), and the genes and corresponding enzymes have been identified (reviewed in [A. Cona, G. Rea, R. Angelini, R. Federico, P. Tavladoraki, Trends in Plant Science 11, 80 (2006)]). Putrescine is converted into GABA through a two-step enzymatic reaction. DAO catalyzes the oxidation of putrescine into gamma-aminobutyricaldehyde, hydrogen peroxide, and ammionium. Gamma-aminobutyricaldehyde spontaneously converts into Δ1-pyrroline. Pyrroline dehydrogenase (PDH) oxidizes Δ1-pyrroline to form GABA. Spermidine is also converted into GABA via PAO to form gamma-aminobutyricaldehyde, which spontaneously converts into Δ1-pyrroline and is converted to GABA by PDH. In bacteria, the catabolism of polyamines into GABA is well documented, and the genes and encoded enzymes have been identified (31, 32). In E. coli, putrescine can be converted into GABA through a two-step enzymatic reaction. An amino group from putrescine is transferred to alpha-ketoglutarate to form gamma-aminobutyricaldehyde and glutamate by putrescine aminotransferase (PAT) (33). Oxidation of gamma-aminobutyricaldehyde by gamma-aminobutyricaldehyde dehydrogenase (GABAlde DeHase) forms GABA (34). Although the polyamine catabolic pathway that forms GABA has been reported in plant tissues (20, 21), there are no reports of the PAT activity in plants, suggesting that plants do not utilize the “bacterial putrescine catabolic” pathway.